1. Field of the Invention
The present invention is directed to forming surface-enhanced Raman spectroscopy (SERS) substrates. In particular, it is directed to flexible SERS substrates with filtering capabilities formed by using nanoparticle-based inks.
2. Description of the Related Technology
Raman spectroscopy (RS) is the measurement of the wavelength and intensity of light scattered inelastically from a molecule. FIG. 1 represents a schematic of the Raman spectrum. Raman scattering is the result of an inelastic collision of a photon with molecules. Both elastic and inelastic collisions occur when light interacts with a molecule. In elastic collisions (Rayleigh scattering), an atom is excited from a ground state to a higher energy state and then relaxes back to the original ground state, thereby emitting a photon at the same frequency as the incident light. However in an inelastic collision, the excited molecule relaxes to a different vibrational state rather than the original state, thereby scattering energy different from that of the incident light. If the scattered energy is higher than the energy of the incident light it is called an Anti-Stokes line (blue shifted), if it is lower it is called a Stokes line (red shifted).
RS gives information about the characteristic vibrational states of molecules. It is a widely used spectroscopic tool for the determination of molecular structure and for compound identification. The Raman scattering signals from the vibrational states of molecules are relatively weak. In order to obtain a satisfactory signal-to-noise ratio, one has to either increase the intensity of the probing laser or resort to surface enhanced Raman spectroscopy. For biological applications, increased laser intensity often limits the in vivo imaging capability of a system. In addition, RS has a small scattering cross section of about 10−30 cm2 per molecule as compared to 10−16 cm2 for fluorescence spectroscopy, thus reducing the possibility of analyzing compounds of biological significance due to the generally low concentration of analytes in biological samples. It is therefore desirable to create SERS structures that permit enhancement of Raman scattering signals for detection of biomolecules.
One such structure employs fractal aggregates of metallic colloidal particles formed on the surface of SERS substrates, typically constructed of Ag, Au, and Cu. Metallic fractal aggregates can exhibit some of the highest SERS signal amplification factors. A fractal is a self-similar geometrical object, i.e. it looks the same at any length scale. Fractal aggregates of metallic colloidal particles can enhance various linear and nonlinear optical responses, including Raman scattering. The basic mechanism that gives rise to such enhancement arises from the localization of optical plasmon excitations within small parts (“hot-spots”) of a fractal aggregate. Such “hot spots” are usually much smaller (tens of nm) than the size of the fractal and often much smaller than the wavelength of the incident light used for detection. Fractal structures, unlike translationally invariant media, cannot support propagating waves and hence can confine electromagnetic fields to very small regions of the substrate. If sufficiently concentrated, the enhanced electromagnetic fields in the hot spots can result in SERS signal amplification.
The regions where the optical excitations are localized have very different local structures and, therefore, are characterized by different resonant frequencies. These nano-scale regions act as a collection of different optical “nano-resonators” resulting in a distribution of resonance frequencies in the visible and infra red spectral ranges and can have resonance quality-factors as large as 103. When Stokes shifts are small, the SERS signal is roughly proportional to the local field raised to the fourth power and, therefore, it can be enhanced up to 1012 in the fractal hot spots.
When two nanoparticles come in close proximity without touching each other, the largest SERS signal amplification is achieved when the analyte molecule is sandwiched between two nanoparticles and when the polarization vector, i.e. the direction of the oscillating E-field of the laser's electromagnetic field, is along the line connecting the centers of the Ag nanoparticles. Amplification factors in the range of 6×106 to 2.5×1010 have been predicted when the separation between two Ag nanoparticles of diameter 90 nm is varied between 5.5 and 1.0 nm. When the polarization vector is perpendicular to the Ag nanoparticles, the maximum amplification factor is relatively small (about 1 to 10). In FIG. 2 the polarization vector with respect to the molecules is shown. The amplification factor for the geometry indicated in [c] is intermediate between cases [a] and [b].
Certain methods of fabricating SERS substrates result in the noble metal nanostructures stochastically distributed over the substrate surface, e.g. electrochemically roughened electrodes, sputtered films, chemically etched films, electroless deposited films, and colloidal metal particles. An exemplary method incubates analyte in an Ag colloidal suspension (in water or other suitable liquid organic carrier) with 1.0-10.0 mM NaCl solution. The role of NaCl is as an aggregating agent. The Ag aggregates are then sorted according to their size and compact aggregates (two to about ten particles each) are isolated for further study using SERS. There are two drawbacks to this technique. First, there is no control over the size of the aggregates produced. The creation of hot spots for Raman scattering largely results as accidental byproducts of the technique. Thus, the reproducibility of SERS substrates made by this method is low. Second, the yield of the desired aggregates is very low and the suitable portions of the nanoparticle array on the substrate must then be selected from a mixture of larger aggregates before they can be used for SERS study. This hinders the fabrication of suitable SERS substrates on a large scale using this method.
Laserna et al. “Surface-enhanced Raman spectrometry on a silver-coated filter paper substrate,” Analytica Chimica Acta, 1988, Vol. 208, pages 21-30, discloses a silver-coated filter paper as a SERS substrate. The filter paper has silver colloidal particles loosely attached to it. The lack of sufficient adhesion to the substrate by the particles causes quick deterioration of the amplification factor over time. In addition, the inter particle spacing is not controlled, but instead is the result of a stochastic process. A similar disclosure is also found in, “Subnanogram Detection of Dyes on Filter Paper by Surface-Enhanced Raman Scattering Spectrometry,” Chieu D., Tran, Anal. Chem., 1984, 56, pp. 824-826.
Another method for fabrication of SERS substrates employs controlled patterning of the nanostructures with electron-beam lithography. One advantage of this method is that little randomness remains and one can expect the SERS signal to be homogeneous across the proposed substrate. Such substrates are now commercially available (Mesophotonics Limited, Southampton, UK). Fabrication of such substrates involves a multi-step process and the resulting substrates are quite expensive. Such substrates are also small in size, for example, typically on the order of 4 mm×4 mm.
Sona et al. “Novel approach for in situ biohazard detection utilizing surface enhanced Raman spectroscopy,” Proc. Of SPIE, 2005, Vol. 5692, pages 351-358, teaches an SERS material made from silver vacuum-evaporated on a fiberglass porous membrane having pore sizes on the order of 1 micron, as well as a chemically deposited thin silver layer on a glass fiber filter. This SERS material was demonstrated for detection of low concentrations of clofibric acid in a liquid and showed the ability to detect the analyte at an extremely low detection level.
U.S. Patent Application publication no. 2007/0259437, discloses nanoparticles coated with a filtering film, which is made from a permselective organic coating agent. The filtering film traps a variety of molecules, but allows the analyte of interest to selectively pass through the film to reach the nanoparticles. RS can then detect the analyte.
U.S. Patent Application publication no. 2006/0060885 discloses a method for depositing a conductive nanoparticle layer onto a substrate surface. A solution of stabilized nanoparticles is applied to a substrate surface and heated to remove liquid vehicle and stabilizer. Heating causes the metal nanoparticles to coalesce to form an electrically conductive layer. Heating is continued until a minimum conductivity of 1 Siemens/centimeter is achieved in order to provide the conductive coating.
The current commercially available SERS substrates have three serious limitations: (a) reproducibility, (b) cost-effectiveness and (c) a small active area (typically 4 mm×4 mm) and the constraint that the analyte has to be brought into intimate contact with the substrate. These limitations make the commercial SERS substrates impractical for detecting molecules of interest in trace amounts or which are dispersed over a large area, e.g. a bio-toxic aerosol released in a large room or a trace amount of bio-hazardous substance released in large volumes of water. In principle it is possible to filter out the molecule(s) of interest and then transfer them to a SERS substrate for further analyses. In practice this is very cumbersome for dilute specimens and therefore is rarely used. The ability to sense molecules of interest at low concentrations is especially critical for effective response to an industrial accident or an act of terrorism or to enhance security at places that may be susceptible to acts of terrorism.
Therefore, there is a need to provide a method for forming SERS substrates that permits control of the average distance between nanoparticles and which provides SES substrates suitable for use to detect trace amounts of analytes in samples of large volumes.